The present invention relates to new types of particles which can be used alone or as vectors for various compounds. It also relates to a process for the preparation of particulate vectors which makes possible improved control of the active principle charging.
Supramolecular Biovectors or SMBV are particles which are biomimetic of the endogenous vectors of the body and which are capable of encapsulating and of carrying a large number of active principles for, in particular, pharmaceutical, cosmetic or agribusiness use.
A first type of SMBV was described in Application EP 344,040. Their structure is very well suited to the role of vector, in particular as a result of the possibility of modifying their size and their composition according to the molecule or molecules transported and their use.
SMBV are synthesized in three successive steps: synthesis of a central core composed, for example, of crosslinked natural polysaccharide, which can be derived by ionic groups and brought, in particular by ultramilling, to the desired size (between 10 nanometers and a few microns, according to the desired use) establishment of a ring of fatty acids grafted covalently solely at the periphery of the central core, in order to confer a peripheral hydrophobic nature on the latter while retaining its internal hydrophilic nature stabilization of one or of a number of external lipid lamellae, composed in particular of phospholipids or of ceramides, sometimes with the addition of other constituents, for example of constituents of biological membranes.
The active principles, according to their physicochemical characteristics, can be transported either in the external lipid lamellae (in the case of lipophilic or amphiphilic compounds) or within the hydrophilic core (in the case of polar compounds).
Encapsulation of active principles of polar nature can take place, according to the structure of the latter, either before formation of the fatty acid ring or between this step and stabilization of the external lamella.
Despite their suitability for many uses, the synthesis of SMBVs can sometimes cause problems and in particular:
it requires a step which is problematic to control in grafting the fatty acid ring;
this grafting, carried out solely at the periphery of the core, must be carried out homogeneously, which requires in particular a prior drying step, under very specific conditions;
if the active principle is encapsulated before the grafting of the fatty acid ring, some of these molecules, localized, after their encapsulation, at the periphery of the core, can be derived by the fatty acid, leading to modification of the properties of this active principle;
if the active principle is encapsulated after the grafting of the fatty acid ring, the latter can be detrimental to the penetration of the active principle into the hydrophilic core.
The present inventors have shown that, surprisingly, in certain applications, it was possible to scale down the reaction scheme by not grafting the ring of fatty acids and phospholipids to the periphery of the crosslinked hydrophilic core.
The present inventors have shown that the polysaccharide particles thus obtained could be used as is. They are then named PS-type SMBV, by analogy with supramolecular Biovector or PSC (polysaccharidic core).
The present inventors have indeed shown that the polysaccharide particles, even of small size, could be used provided that suitable charging protocols are adopted.
This is why the subject of the present invention is a synthetic particulate vector, characterized in that it comprises:
a non-liquid hydrophilic core.
A further subject of the present invention is a synthetic particulate vector which consists essentially of a non-liquid hydrophilic core.
The notion of vector must, in this instance, be understood within the broad meaning, that is to say that it comprises particles having a support role, for example when they are incorporated in a composition, either as such or for the transportation, the presentation and/or the stabilization of active compounds.
A non-liquid hydrophilic core (or matrix) can be a hydrophilic polymer. The hydrophilic matrix can in particular be composed of polysaccharides or oligosaccharides which are naturally or chemically crosslinked. The polysaccharide is preferably chosen from dextran, starch, cellulose and their derivatives.
The hydrophilic core can be obtained by various methods and in particular, if it is a core of polysaccharide nature, by using a branched or linear biodegradable polysaccharide. This polysaccharide can be, for example, starch or one of its derivatives. Crosslinking processes are known to a person skilled in the art and can be carried out by means of bi- or tri-functional agents, such as epichlorohydrin or phosphorus oxychloride.
The properties of the polysaccharide can be modified by grafting the sugars by acidic or basic ionic functional groups which are important for the encapsulation of ionic active principles.
Encapsulation of the hydrophilic active principles can be carried out at this stage of the synthesis. The gel obtained during the synthetic step is then washed and partially dehydrated by means, for example, of centrifugation techniques, then brought into the presence of the active principle and slowly rehydrated. As the gel has the ability to swell with water, the active principle is carried within the polysaccharide network where it can be bound by ionic bonds with the groups grafted within the gel.
The gel obtained, whether it contains or does not contain an encapsulated compound, must be mechanically ground for the purpose of obtaining particles of desired size. The ultramilling methods are known in the state of the art and can in particular involve a high pressure extrusion using a homogenizer.
Another subject of the present invention is a process for the preparation of a particulate vector, comprising:
a) encapsulating a basic ionizable active principle in a crosslinked hydrophilic matrix grafted by acidic ionic ligands, at a pH below the pKa of the active principle; and
b) increasing the pH of the medium to a value above the pKa of the active principle.
In fact, the adoption of a suitable protocol for the charging of hydrophilic cores makes it possible to control the topology of the charging.
The hydrophilic matrix is preferably composed of polysaccharides or of oligosaccharides, which are naturally or chemically crosslinked.
This process, which can be used with SMBV, is more particularly important with particles in which the external lipid lamellae have been reduced (L-type SMBV) or eliminated (PS-type SMBV) with respect to the method described above. The present inventors have observed that it is difficult to use such SMBV containing reduced lipid lamellae as vectors for the encapsulation of ionic active principles with conventional charging methods.
In fact, if molecules of the active principle are bound with the polysaccharide particle of the core while being maintained at the periphery of the core, this can result in an instability in the particle suspension, it being possible for the particles to aggregate with one another by virtue of interparticulate bonds due to the active principle. This phenomenon is relatively minor for low levels of charging of active principles, whereas it becomes very important with high levels of charging of active principles. Likewise, the size of the particles is extremely important. With particles of large size (for example, greater than 100 manometers), the ratio of the surface area to the internal volume of the particle is very low; for this reason, in comparison with the total amount of active principle encapsulated, the amount of active principle bound at the periphery of the particles is very low, thus limiting the possibilities of interparticulate bonds. In contrast, when the particles are very small in size, this aggregation phenomenon is very noticeable. It should also be noted that this phenomenon is not very marked with SMBV having a layer of fatty acids grafted onto the core, which then serves to isolate from interparticulate interactions.
In order to overcome this, the present inventors have shown that it is possible to control topologically the penetration of the active principle within the particles by controlling the ionic conditions of the encapsulation.
The polysaccharide cores can be regarded as polyelectrolyte matrices and, as such, they have a pH differential between the inside and the outside of the particle. This phenomenon is due to the more or less significant dissociation of the counterions and to the immobilization of the ionic functional groups on the polysaccharide network. This property makes it possible to control the localization of the active principle to be incorporated, by causing a solely internal encapsulation or an encapsulation solely at the surface or alternatively in the periphery of the core.
When the active principle to be incorporated is basic in nature, its solubilization in a medium with a pH below its pKa leads it to exist in the ionized form; it can then become attached to the anionic groups grafted onto the polysaccharide core. When the pH rises above the pKa, the active principle is in a deionized form, which does not allow it to interact with the matrix. In order to control the localization of the encapsulated active principle, use is therefore made of the pH differential which exists between the inside and the outside of the particle: if the external medium has an excessively high pH, the active principle cannot interact with the ionic groups placed at the periphery of the cores. The internal pH of the cores derived by acidic ligands being lower than the external pH, the active principle, which has entered the particle in the deionized form, becomes ionic again and thus is bound to the anionic groups of the L-type SMBV. In this specific case, the active principle will be localized solely in the core of the particle, to the exclusion of the peripheral region. This type of encapsulation is thus very favorable to an optimum dispersion of the particles.
In the case of acidic active principles, it is possible symmetrically to apply the process with cores derived by basic ligands, according to the following steps:
a) encapsulating an acidic ionizable active principle in a crosslinked hydrophilic matrix grafted by basic ionic ligands, at a pH above the pKa of the active principle; and
b) decreasing the pH of the medium to a value below the pKa of the active principle.
This type of charging with topological control of the localization of the active principle in the polysaccharide core is particularly advantageous for vectorization applications with SMBV in which the external lipid lamellae have been reduced (L-type SMBV) or eliminated (PS-type SMBV) but it is also suitable and desirable for SMBV already described in the above patents, in order to increase the degree of charging or to minimize the disturbances caused to the structure of the external phospholipid lamella in the case of external attachment of macromolecules, for example of recognition units and in particular of an apoprotein.
Another subject of the invention is a particulate vector composed of a crosslinked hydrophilic core grafted by ionic groups and here called PS-type SMBV. The ionic groups can be anionic groups, such as for example phosphates, succinates or carboxymethylates, or cationic groups, for example quaternary ammoniums or amines. The size of the PS-type SMBV is preferably between 20 and 200 nm.
The crosslinked hydrophilic core can be composed of natural or synthetic polymers which are naturally or chemically crosslinked. Use is in particular made of polysaccharides or oligosaccharides, such as starch, dextran, cellulose and their derivatives.
Advantageously, an active principle is encapsulated in the PS-type SMBV mainly at the center of the matrix; the external part of the core is virtually devoid of active principle, which makes it possible to avoid the aggregation phenomena which generally occur for particles of small size.
One of the subjects of the invention is therefore a particulate vector composed of a crosslinked polysaccharide matrix containing an active principle, the active principle preferably being localized mainly at the center of the matrix.
According to yet another aspect, a subject of the invention is a process for charging which makes it possible to encapsulate the active principle in a complete SMBV, a PS-type SMBV or an L-type SMBV, which can be in the form of a suspension.
The charging is carried out on the particulate vector. In order to do this, the hydrophilic core must contain ionic groups. The process thus requires the following steps:
a) a crosslinked hydrophilic core is prepared in which ionic groups are fixed,
b) the active principle is charged within the vector at a pH suitable for the active principle and while supplying energy,
c) having incorporated the active principle, the vector is recovered.
In the case of PS-type SMBV, it is difficult to use conventional charging methods for incorporating ionic active principles. It is true that methods with topological control make it possible to overcome this problem. However, topological control requires precise adjustment of the pH which must be compatible with the active principle and the vector.
The present inventors have, therefore, developed an alternative method to overcome these problems. The ionic ligands grafted into the polysaccharide network of the vectors result in a significant affinity for the ionic active principles of opposite charge. However, this affinity, during the incorporation, must be controlled in order to avoid aggregation of the vectors and to make it possible to localize the active principle mainly within the particles. For the precharging, this control requires precise adjustment of the pH or a low level of incorporation. This aggregation is mostly due to localization of the active principle at the surface, which localization is itself due to the presence of ligands at the surface of the particles.
In order to effect this new type of charging, three factors come into play:
a) a significant affinity of the active principle for the vector in order to provide for incorporation of the active principle: this affinity is created by acidic or basic ionic ligands which are grafted into the crosslinked polymer; the density and the strength of the ligands can be adjusted according to the active principle,
b) a significant dispersion of the vectors during the incorporation in order to avoid the interactions between particles which promote aggregation: this dispersion can be provided for by the dilution of the vectors in the reaction medium at a concentration which is sufficient to decrease the interparticulate interactions but also at a concentration which is compatible with pharmaceutical applications,
c) the use of any means for promoting entry of the active principle within the vector: the contribution of energy, in the form, for example, of stirring or of heat, will accelerate the kinetics of entry of the active principle but will also promote dispersion of the vectors; the appropriate form of the active principle, which must be sufficiently ionic to make it possible to attach the active principle but also the least charged, in order to avoid surface interactions.
For SMBV or L-type or PS-type SMBV, it is possible to use incorporation protocols corresponding to these requirements. The presence of grafted ionic ligands in the crosslinked polymer provides for attachment of the active principles for the three species. Dispersion of the vectors can be carried out by suspending PS-type SMBV in water. SMBV or L-type SMBV are prepared from acylated or polysaccharide cores and from phospholipids dispersed beforehand in aqueous medium and are thus suspended in water. The contribution of energy, for example, in the form of stirring or of heat, does not damage the SMBV. It is possible to vary the pHs and to define pH ranges which are compatible with this type of charging.
This new process makes it possible to prepare SMBV of any type which are charged with active principle, while retaining the size of the base vectors. This process thus has many advantages. This method comprises preparing the blank vectors, without active principle, before the incorporation. This makes it possible to process the blank vectors according to conditions which are suitable for the vectors and which do not depend on the active principle to be encapsulated. The vectors are subsequently charged. These conditions can, therefore, be more or less drastic. They also make it possible to be able to characterize the blank vector as a base entity.
Incorporating the active principle in the final step of the process results in the active principle, which is capable of being toxic and expensive, being handled during only one step of the process. This process thus reduces the handlings and the possible losses of the active principle. It, therefore, makes it possible to be more certain as regards safety, but also more profitable.
In addition, for some active principles, the incorporation conditions can be relatively simple, which makes it possible to envision charging the vectors with the active principle at the time of use. This method of preparation at the time of use can eliminate the problems of storage in the liquid state.
This new method of charging is based on the significant affinity between the vectors and the ionic active principles, but also on the simple control of the incorporation by the dispersion of the vectors and the ionic form of the active principle. It has very worthwhile advantages: preparation of the blank vector independent of the active principle, handling of the active principle in a single step and the possibility of preparation at the time of use.
The particulate vectors according to the invention preferably have a diameter of between 10 nm and 5 xcexcm and more preferably between 20 and 70 nm.
These particulate vectors are intended to carry or to present at their surface one or a number of molecules possessing biological activity. Mention must be made, among these molecules, without this list being limiting, of:
antibiotics and antivirals,
proteins, proteoglycans, peptides,
polysaccharides, lipopolysaccharides,
antibodies,
antigens,
insecticides and fungicides,
compounds which act on the cardiovascular system,
anticancers,
antimalarials,
antiasthmatics,
compounds having an effect on the skin,
constituents of dairy fat globules.
In the examples below, a description will be given of the charging of various products according to their characteristics, and in particular:
a hydrophilic product of small size intended for systemic administration,
an active principle possessing anticancer activity,
two enzymes possessing antibacterial activity, lactoperoxidase and glucose oxidase, and
a plant extract composed of procyanidol oligomers possessing an antioxidant activity,
constituents of the fat globule of milk.
The present invention, therefore, provides a pharmaceutical composition, comprising a particulate vector according to the invention and a pharmaceutically acceptable support for its administration. The vectors according to the invention are in particular useful for therapeutic and immunological applications.
The present invention also provides a cosmetological composition comprising a particulate vector as described above, and cosmetologically acceptable excipients.
Finally, food compositions comprising particulate vectors according to the invention form parts of the invention.
The examples which follow are intended to illustrate the invention without limiting the scope thereof.